人骨髓间充质干细胞的生物学特性分析

Isolation and cultivation of human bone marrow mesenchymal stem cells in vitro andanalysis of their biological characterizations

目的对人骨髓间充质干细胞（HMSCs）的生物学特性进行初步分析，为其临床应用奠定基础。方法抽取4例健康人髋骨内骨髓5～20ml，密度梯度离心法分离HMSCs。体外培养扩增后流式细胞仪检测HMSCs表面标记物；MMT法绘制生长曲线，计算细胞倍增时间；吉姆萨染色法检测细胞核型。结果HMSCs表面标记物CD29、CD44、CD71、CD105、CD166、HLA-ABC、UEA-1阳性表达；CD34、CD45、HLA-DR阴性表达；生长曲线呈S形，细胞倍增时间随代次增加而延长；核型分析显示第3、6代HMSCs的染色体数目未发生改变。结论密度梯度离心法可得到纯度较高的HMSCs，表可达人类间充质干细胞的表型特征。HMSCs可在体外较长时期培养。在第7代之前细胞增殖旺盛，之后细胞增殖能力逐渐下降；在第6代之前染色体数目未发生改变。

[Objective] To establish a simple and feasible method to culture and proliferate human bone marrow derived mesenchymal stem cells （HMSCs） in vitro and analyze their biological characterizations. [Methods] HMSCs were isolated by combining gradient density centrifugation with plastic adherence. The growth curves of HMSCs were drawn by MTT assay, cell phenotypes were detected by flow cytometry, cell doubling time was caculated, and cell karyotypes were measured by Giemsa staining. [Results] The positive expression of cell phenotype of HMSCs includes CD29, CD44 CD71 CD105, CD166 HLA-ABC, UEA-1, while the negative expression of cell phenotype includes CD34, CD45, HLA-DR. The growth curve of HMSCs was ＂S＂ shaped. The cell doubling time prolonged with the passages increased. The chromosome number of HMSCs of passage 3 and 6 were both 46, same as human beings. [Conclusion] I-LMSCs of higher purity can be isolated by combining gradient density centrifugation with plastic adherence, and maintain cell phenotypes as human beings. The proliferation ability of HMSCs is strong before passage 7, but decreases rapidly later. The chromosome number of HMSCs remains original before passage 6.